EP3856414A1

EP3856414A1 - Microfluidic method for analysing metals

Info

Publication number: EP3856414A1
Application number: EP19773850.3A
Authority: EP
Inventors: Floriant DOUNGMENE; Lisa GAUTRIN; Aurélie MAGNIER; Clément NANTEUIL
Original assignee: Klearia SAS
Current assignee: Klearia SAS
Priority date: 2018-09-28
Filing date: 2019-09-27
Publication date: 2021-08-04
Also published as: FR3086759A1; WO2020064987A1; US20220032297A1

Abstract

The present invention relates to a microfluidic method for analysing a fluid containing a metal trace element, in particular arsenic, comprising the following steps: a) introducing a fluid sample into at least one micro-channel of a microfluidic circuit; b) mixing, in the micro-channel of the microfluidic circuit, the introduced fluid sample with nitric acid and L-cysteine; and c) measuring the quantity of metal trace element present in the sample using an electrochemical detection method.

Description

Microfluidic metal analysis process

The present invention relates to a method for analyzing a fluid containing a metallic trace element, for example arsenic, using a microfluidic method. The invention also relates to a microfluidic circuit, allowing the simple, automated handling, of very small quantities of fluids, making it possible in particular to implement such a method.

Arsenic is a natural element of the earth's crust that is widely present in the environment, whether in the air, in water or in the earth. It is very toxic in inorganic form.

People are exposed to high levels of inorganic arsenic by drinking contaminated water, using it to prepare meals, irrigating crops, during industrial processes, eating contaminated food or smoking tobacco . Prolonged exposure to inorganic arsenic, mainly by drinking contaminated water or eating foods prepared with this water or from crops irrigated with arsenic-rich waters, can lead to chronic poisoning. Skin lesions and cancers are the most characteristic effects. Other adverse health effects that may be associated with prolonged ingestion of inorganic arsenic are: developmental effects, neurotoxicity, diabetes, pulmonary and cardiovascular disease. In particular, arsenic-induced myocardial infarction can be a major cause of excess mortality (see WHO website).

The greatest public health threat from arsenic comes from contaminated groundwater. Inorganic arsenic occurs naturally at high levels in groundwater in a number of countries, including Argentina, Bangladesh, Chile, China, India, and Mexico (see WHO website).

In its Drinking Water Quality Guidelines, WHO estimates that the currently recommended limit for arsenic in drinking water is 10 pg / liter, although this value is provisional due to dosing difficulties and practical complications for removing it from drinking water. Conventionally, the determination of arsenic in drinking water is carried out by samples, which are transmitted and analyzed in the laboratory. Consequently, this type of test requires multiple manipulations of the sample (especially during transport, packaging, etc.), at the risk of modifying its composition. Furthermore, such tests take a long time to implement (at least a few days), before obtaining the results.

The present invention aims to overcome these drawbacks of the prior art. In particular, the present invention aims to propose a method for analyzing a fluid containing a metallic trace element (ETM), in particular arsenic, using a microfluidic method, which is simple and quick to implement. work, but also reliable, having a very good sensitivity and whose measurements are reproducible. In particular, the method according to the invention does not distort the sample tested, and / or avoids the hydrolysis of the ETMs to be analyzed. The detection limit of the species to be analyzed is compatible with the regulatory thresholds (pg / L or less). Finally, it requires a very small sample volume.

Another objective of the present invention is to provide a microfluidic circuit allowing the implementation of such a method.

The invention thus relates to a microfluidic method for analyzing a fluid containing at least one metallic trace element comprising the following steps:

a) introduction of a fluid sample into at least one microchannel of a microfluidic circuit;

b) mixing, within the microchannel of the microfluidic circuit, of the sample of fluid introduced in step a), with reagents, and

c) measurement of the quantity of metallic trace element present in the sample obtained in b), using an electrochemical detection method.

Preferably, step c) is carried out using at least 2 electrodes, preferably at least 3 electrodes, preferably at least 3 electrodes, one of which is made of gold. Preferably, step c) is carried out using at least one platinum electrode as the reference electrode. Preferably, the measurement in step c) is carried out using the following three electrodes:

- a gold electrode, as a working electrode,

- a platinum electrode, as a reference electrode, and

- a platinum counter electrode. Preferably, step c) comprises mixing the sample obtained in b) with at least one solution comprising a known concentration of metallic trace element ("standard solution"), then the assay of the metallic trace element by electrochemical detection method.

Preferably, the invention relates to a microfluidic method for analyzing a fluid containing arsenic, comprising the following steps:

b) mixing, within the microchannel of the microfluidic circuit, of the fluid sample introduced in step a), with nitric acid and L-cysteine, and

c) measurement of the amount of arsenic present in the sample obtained in b), using an electrochemical detection method.

Preferably, said step c) comprising the mixing of the sample obtained in b) with at least one solution comprising a known concentration of metallic trace element, then the assay of the metallic trace element by electrochemical detection method.

Preferably, step c) is carried out using at least 2 electrodes, preferably at least 3 electrodes, preferably at least 3 electrodes, one of which is made of gold.

The electrode can be any electrode usable in electrochemistry, such as an electrode made of gold, possibly covered with gold nanoparticles; a thin layer electrode; or a carbon nanotube electrode.

This process can be implemented particularly easily, in a single microfluidic circuit in which the various steps are carried out. This circuit is illustrated in the detailed figure below.

The method according to the invention is preferably fully automated, and allows the user to dispense with the different stages of pre-treatment of the sample and the stages of analysis which are sometimes complex and require the handling of chemicals such as concentrated acids or ETM standard solutions to be analyzed.

By “fluid” is meant any liquid body capable of assuming the shape of the container which contains it. Preferably, the fluid according to the invention is a solution. Preferably, the fluid according to the invention is water. The water tested according to the method according to the invention can be any type of water. By “metallic trace element”, also called “ETM”, is meant a toxic or toxic metal beyond a certain threshold. Preferably, IΈTM is chosen from lead, mercury, arsenic, copper, zinc and cadmium. Preferably, IΈTM according to the invention is arsenic.

Preferably, the method according to the invention is a method for analyzing water containing an ETM, in particular arsenic.

Preferably, it is implemented using a portable device including the microfluidic circuit.

The method according to the invention can also be used for the analysis of any ETM present in a solution or a trace fluid.

The use of nitric acid as reagent used in the method according to the invention, in particular for the detection of arsenic, is particularly relevant: in fact, the analysis of trace ETMs like arsenic is most often done in an acid medium to minimize interference, to improve sensitivity and to avoid hydrolysis of the ETMs to be analyzed or of the species formed after electrochemical transformation (D. Jagner, M. Josefson, S. Westerlung, Anal. Chem. 53 (1981) 2144; J. Lexa, K. Stulik, Talanta 30 (1983) 845 and E. Munoz, S. Palmero, Talanta 65 (2005) 613-620). Hydrochloric acid (HCl) is the most used because it leads to the best detection limits (in English "level of detection" or LOD) (E. Munoz et al supra). However, this acid is known to attack the gold electrodes used to measure the amount of ETM, and therefore reduce the lifetime of the analysis system (E. Munoz et al supra).

Another acid could also be used, such as in particular hydrochloric acid, sulfuric acid or acetic acid.

However, advantageously, the use of nitric acid, in combination with L-cysteine and the confined nature of the reagents in the microfluidic channels (reaction and rapid diffusion of the species), has made it possible to obtain LODs comparable to those obtained with hydrochloric acid. In addition, unlike hydrochloric acid, the use of nitric acid makes it possible to carry out a very large number of analyzes, ie at least 400, preferably at least 450, preferably at least 500 analyzes, without destroying the 'gold electrode. Indeed, the advantage of the microfluidic circuit according to the present invention lies in the fact that it is reusable, and not for single use; it typically allows at least 400 analyzes, preferably at least 450, preferably at least 500 analyzes, without destruction of the gold electrode.

L-cysteine is a reagent that converts arsenic (V) (AsV) to arsenic (III) (Aslll) when the solution is heated. It therefore allows the speciation of arsenic, ie to differentiate Aslll and AsV. The transformation reaction is given below (Talanta 58 (2002) 45-55):

- CH {-NH 1 COOH (cystinc) - + - H ₂ 0

or in a simplified way: AsV + cysteine => Aslll + cystine.

Preferably, the method according to the invention does not use hydrazine hydrochloride (N2H4-HCl).

Preferably, step c) of the method comprises mixing the sample obtained in b) with at least one solution comprising a known concentration of metallic trace element ("standard solution"), then the assay of the element - metallic trace by electrochemical detection method.

Preferably, at least one standard solution is used, preferably at least two standard solutions. By "standard solution" is meant an ETM solution whose concentration is known. This concentration is determined upstream as a function of the concentration of ETM to be analyzed in the sample.

Typically, when the IΈTM to be measured is in a low concentration, in particular strictly less than 10 ppb ("low range"), two standard solutions are used, for example at 2 and 4 ppb of ETM.

Typically, when the IΈTM to be measured is in a higher concentration, in particular greater than or equal to 10 ppb (“high range”), for example between 10 and 20 ppb, two standard solutions are used, for example at 10 and 20 ppb of AND M.

The use of at least one standard solution according to the invention makes it possible to dispense with matrix effects. The microfluidic method according to the invention is thus reliable and reproducible. In addition, it can be placed on any type of fluid, and does not require no prior calibration. Such a method is thus based on metered additions of ETM in the sample.

According to an alternative embodiment, it is possible to set the discriminating value between the low range and the high range with respect to the regulatory value of IΈTM to be measured. When the concentration of ETM in the sample is measurable in the low range, it is not necessary to obtain its specific concentration: the simple fact of being able to measure the concentration of ETM in the low range means that the latter is less than the discriminating value, and therefore the regulatory value.

According to a first embodiment, the method according to the invention can comprise, in step c), the detection of IΈTM in the high range or in the low range. According to this first mode, typically, the concentration of IΈTM is not precisely measured. The assay then only includes the detection of IΈTM in the high range (range of concentrations greater than or equal to 10 ppb) or in the low range (range of concentrations less than 10 ppb).

According to a second embodiment, the method according to the invention may comprise, in step c), the detection of IΈTM in the high range or in the low range, then the measurement of concentration of IΈTM within this range. The assay then comprises the detection of IΈTM in the high range or in the low range, then the measurement of concentration of IΈTM within the range.

The invention also relates to a microfluidic circuit for analyzing a fluid, in particular capable of implementing the method according to the invention, comprising:

- at least one reagent storage tank (s), preferably nitric acid and L-cysteine, and optionally at least one second storage tank comprising at least one standard solution;

- at least a first microfluidic chip, called a premix chip, comprising at least a first microchannel fluidically connected:

o at a first end, two tanks and an inlet, and o at the second end, a tank,

said inlet suitable for injecting a sample of fluid to be analyzed; and

- at least a second microfluidic chip, called an analysis chip, comprising at least a second microchannel connected to the tank and comprising at least two electrodes, preferably at least three electrodes, one of which is made of gold.

The first chip and the second chip according to the invention can also, according to one embodiment, be prepared on a single and same substrate. In such a case, we get thus a single chip, comprising a first compartment (corresponding to the premix chip) and a second compartment (corresponding to the analysis chip).

Preferably, the microfluidic circuit comprises, in the first chip, a first network of microchannels in which the sample of fluid to be analyzed and the reagents (such as nitric acid and L-cysteine) circulate; and in the second chip, a second network of microchannels comprising at least 2 electrodes, preferably at least three electrodes.

Preferably, the first network of microchannels is present in a first microfluidic chip, generally called “pre-treatment chip” or “mixing chip”; it is used to mix the various reagents (such as nitric acid and L-cysteine) with the sample, in particular in specific proportions.

Preferably, the second network of microchannels is present in a second microfluidic chip, generally called "analysis chip"; it is the chip on which the target pollutants are detected and quantified, thanks to the presence of two or three electrodes, and in particular also thanks to the presence of calibration solutions (or standard solutions).

The circuit according to the invention, integrated in particular in chips as described above, allows the implementation of the method described above, in a particularly easy way.

Microfluidic chips, also called "labs on a chip" or "lab-on-a-chip" according to the English terminology sometimes used by those skilled in the art, are miniaturized devices for biological or chemical analysis, which consist of at least one thin plate (of the order of a few tens to a few hundred micrometers), preferably made of glass (that is to say a hard, brittle and transparent substance, of vitreous structure, essentially formed of alkaline silicates, and having a high chemical resistance), and of a cover comprising at least one microfluidic channel (or microchannel). Each chip is preferably as described in EP2576056.

Preferably, the chips constituting the microfluidic circuit according to the invention (i.e. pre-treatment chip and analysis chip) each include:

- a plate,

a cover comprising at least one microfluidic channel, and - a single layer, intermediate between the plate and the cover, formed of an inorganic matrix of Si02.

Preferably, the single layer has a thickness between 100 nm and 10 mm, and preferably between 300 nm and 400 nm. Preferably, each chip comprises at least one circuit in the cover and / or at least one circuit on the cover, associated with the cover by an inorganic matrix of SiO 2.

Preferably, the microfluidic chips according to the invention are made of glass. In fact, when the plate and the cover of said chip are made of glass, the chips fully benefit from the exceptional properties of glass, namely:

- high optical transparency allowing good observations;

- high mechanical strength, with a high Young's modulus and a high breaking stress (depending on the type of glass used);

a low porosity, which makes the chip perfectly suitable for chemical analysis applications under very low concentration conditions (analysis of weakly concentrated toxins for example) which avoids any pollution from the outside of the chip and any leakage of dangerous products to the outside;

- chemical inertness to most chemical compounds (with the exception of hydrofluoric acid derivatives), for example such as concentrated acids and non-aqueous solvents. Various chemical solutions can therefore be circulated in the microchannels. For example, the surfaces of the fluid channels are naturally hydrophilic following the chemical treatments carried out for the manufacture of the chip. This specificity is important for the analysis of biological samples. However, if necessary, it is also possible to treat the fluid channels to make them hydrophobic, by circulating a suitable solution. Finally, the chip can undergo chemical cleaning, and can be biopassivated on the surface by a simple circulation of suitable liquid known to the skilled person, to obtain biocompatibility at will. We see here the advantage of the chemical inertness of the chip;

- a significant electrical insulation which allows in particular a correct functioning of the circuits and the application of strong external electric fields (as in the case of capillary electrophoresis on a chip for example).

The present invention will be better understood on reading the following description of preferred embodiments, given by way of illustration and not limitation, and accompanied by the attached figure, which is a plan, in top view, of a circuit comprising microfluidic chips allowing the implementation of a method according to the invention. Microfluidial circuit

FIG. 1 is a plan, seen from above, of a circuit comprising the microfluidic chips making it possible to implement a method according to a preferred embodiment of the invention. Preferably, this circuit comprising the microfluidic chips is suitable for the detection of arsenic in a sample of fluid such as water. This plan shows the different microfluidic channels which are formed inside this circuit.

This circuit generally consists of the following elements:

- the reservoirs (R.3, R.4, R.5, R.7 and R.8 to R.13): their role is to store the different reagent solutions and the sample to be analyzed.

In particular, tank R.3 contains the sample to be analyzed.

Typically, the tank R.4 is used to store nitric acid (HN0 ₃ ), preferably with a concentration of 2.2 M. Nitric acid has a double role: as indicated above, it makes it possible to clean the circuit microfluidic, but also to acidify the sample to be analyzed.

Preferably, the circuit comprises a reservoir R.5, which contains a mixture of nitric acid (especially at 100 mM) and L-cysteine (especially at 15 mM): it serves as a measurement blank, or control solution. In other words, tank R.5 contains a sample devoid of ETM, which serves as a control. It verifies that the circuit has not been contaminated by the sample to be analyzed.

Reservoir R.7 contains L-cysteine, preferably at 50 mM.

Preferably, the tanks R.8 to R.1 1 contain calibration solutions (or standard solutions). In particular, they contain solutions of As (lll) with respective concentrations equal to 14.48, 28.96, 72.40 and 144.80 ppb, acidified with 10 mM nitric acid. These solutions are used to add 2 and 4 ppb of As (lll), or 10 and 20 ppb of As (lll), to the sample to be analyzed (by definition of unknown concentration), which depends on the concentration to be analyzed. For a range of concentrations to be analyzed between 0 and 10 ppb, the additions of the solutions at 2 and 4 ppb are used, while the additions of the solutions at 10 and 20 ppb are used for concentrations greater than 10 ppb.

Preferably, the reservoir R.12 contains an aqueous solution of sulfuric acid (H ₂ SO4), preferably at 100 mM. It allows in particular the cleaning of the working electrode of the analysis chip, electrochemically between different measurements. Finally, preferably, the reservoir R.13 contains a mixture of tetrachloroauric acid (HAuCL), preferably at 2 mM, and sulfuric acid (H ₂ SO4), preferably at 100 mM. This solution is used to automatically regenerate the working electrode (in particular gold) of the analysis chip by electrochemical means, in the event that its surface deteriorates.

- the internal (PI) and external (PE) bins:

The external bin contains the excess sample to be analyzed which has been injected into the system, or which has been used to rinse the tank R.3.

The internal bin (bin with gas permeable cap), inaccessible to the user, contains all the solutions containing chemicals, such as acid solutions, L-cysteine solution or even the mixture of tetrachloroauric acid and sulfuric acid.

- the tank (C):

It is a recirculation tank with an inlet and an outlet. It is used to heat the sample to be analyzed, in the presence of L-cysteine, and thus carry out the conversion of As (V) into As (III). This heating is typically carried out by means of a heating resistor mounted on the tank.

- the bubbles (references 8 and 10 in FIG. 1):

The bubbles are indicated in a circle in the figure. There is one between the tank R.3 and the solenoid valve 1 (spike 8); and another at the outlet of the solenoid valve 14 (spike 10). They remove air or gas bubbles trapped in the liquid circulating in the circuit.

- the solenoid valves (EVs), represented by crosses in the figure:

These are electrically controlled systems, which allow the passage or not of the liquid in the circuit. Thus, when a solenoid valve is open, it will allow the liquid to pass through, while when it is closed the liquid is blocked and cannot pass.

- the connection elements (tubes, screws, unions, ferrules), represented by an hourglass as reference 22 in Figure 1:

These are the different elements that connect the different parts of the circuit. The liquid can thus circulate throughout the circuit. - a gas cylinder (G):

A 12-liter bottle of nitrogen is used to generate pressure to move the different solutions through the system. Preferably, a positive pressure of 500 mbar is used throughout the analyzes. This cylinder is connected to the circuit by means of the solenoid valve 3, which allows the injection of gas into the circuit.

- microfluidic chips (50 and 60):

o pretreatment chip or mixing chip (reference 50 in FIG. 1): It allows a pretreatment of the sample, by carrying out the various mixtures in the desired proportions. The user is therefore freed from these steps which are often long and require the handling of dangerous reagents, such as concentrated acids.

It is delimited, in the figure, by the inputs 51 to 58, the input 51 being connected to the solenoid valve 21; and by the output of the main microchannel 59 (also called “first microchannel”) on the solenoid valve 12. The main microchannel 59, also called “first microchannel”, is the microchannel starting at the input 51 and ending at the solenoid valve 12.

More specifically, the inlet 51 is connected to the outer trash PE, via the solenoid valve 21. The inlet 52 is connected to the tank R.4 (containing the nitric acid) via the solenoid valve 7.

Inputs 53, 56 and 58 are not linked. They can be provided in particular for the following cases: an entry is provided for the addition of a complexing agent, such as EDTA, to complex potential metallic interferers; another inlet is provided for diluting the sample with water, in particular ultrapure, if it is very concentrated and falls outside the linearity range of the sensor; finally the last entry is suitable for a more concentrated nitric acid solution, which would be used in the event of dilution, in order to bring the final pH to an acceptable value, for example 1.

Input 54 is connected to reservoir R.3 (containing the sample to be analyzed) via solenoid valve 1.

Input 55 is connected to tank R.4 (containing nitric acid) via solenoid valve 6. Finally, input 57 is connected to tank R.7 (containing L-cysteine) via solenoid valve 4.

This chip 50 has a channel depth of 50 µm. Typically, the length between the inlet 51 and the outlet 59 is 142 mm. The main microchannel 59 has a width typically between 0.7 and 2 mm, preferably between 0.7 and 1.5 mm, preferably equal to 1 mm.

Preferably, the flow rate of the main microchannel is 11 ml / h for a pressure of 500 mbar.

Preferably, the dimensions of the different channels of the preprocessing chip are as follows:

Preferably, the dimensions of the different channels are chosen so that at the outlet of the main microchannel of the chip, the mixtures are homogeneous and in the proportions below:

Preferably, the dimensions of the mixing chip 50 are such that for a normalized total volume leaving the chip (equal to 1), the volume of sample to be analyzed is 0.63, the volume of nitric acid is 0.04 and the volume of L-cysteine is 0.32. In this case, we have a dilution of the sample of 1.58 (= 1 / 0.63). o analysis chip 60:

This chip 60 comprises a system of three electrodes, on which the mixtures leaving the pretreatment chip 50 are analyzed.

The analysis chip 60 is delimited, in the figure, by the inputs 61 to 67, the input 61 being connected to a spike 10 and to the solenoid valve 14, by the inputs 70 to 75; and by the exit of the main microchannel 69 (also called “second microchannel”) in the internal trash PI.

The main microchannel 69 has a width typically between 0.7 and 2 mm, preferably between 0.7 and 1.5 mm, preferably equal to 1 mm.

The analysis chip 60 typically has 13 inputs (references 61 to 67 and 70 to 75) and an output 76. Among these inputs, there are the inputs 70 to 75 connected to the tanks R.8 to R.13, and the entries 61 to 67.

The inlet 61 is connected to a bubble separator 10, to the solenoid valve 14 and to the tank R.4 (containing the nitric acid) via the solenoid valve 20.

The input 62 is connected to the tank R.5 (containing the measurement blank) via the solenoid valve 17.

Inputs 63 to 67 can be connected to ultra pure water tanks, and / or for the addition of buffer solutions, such as acetates or phosphates. They allow measurements to be made on highly concentrated samples, or at pH levels close to the pH of drinking water, therefore without acidification.

In addition, the tanks R.8 to R.1 1 (containing the calibration solutions) are connected to the main microchannel 69 via, respectively, the solenoid valves 26, 28, 27 and 25.

The tank R.12 (containing the aqueous sulfuric acid solution) is connected to the main microchannel 69 via the solenoid valve 18.

Finally, the reservoir R.13 (containing the mixture of tetrachloroauric acid and sulfuric acid) is connected to the main microchannel 69 via the solenoid valve 24.

In detail, preferably, the system of three electrodes comprises:

a 1.06 mm x 1 mm dimension working gold electrode,

a platinum reference electrode of 2.96 mm x 1 mm dimension, and a platinum counter electrode of 6.74 mm x 1 mm dimension,

all the electrodes being located in the main microchannel 69 (not shown in FIG. 1). The depth (or width) of the analysis chip 60 is 20 µm. The length between the inlet 61 and the outlet 76 is typically 178 mm, and the width of the main microchannel 69 is 1 mm.

Preferably, the average output flow is around 400 mI / h at a pressure of 500 mbar.

Typically, the sample coming from the pretreatment chip enters through one of the channels of this chip, for example input 61, just like the nitric acid of the reservoir R.4.

Another channel (ie the inlet 62 via the solenoid valve 17) is dedicated to the measurement blank (tank R.5) (control solution), which serves as a control to ensure that there is no contamination from the system.

The dimensions of the different channels of the analysis chip necessary to obtain the ratios allowing a satisfactory LOD are the following:

Typically, each chip (pretreatment chip and / or analysis chip) can be composed of two superposed plates, glued to one another. Thus, each chip can be composed of a first plate, which can for example be a transparent blade microscope, and a second plate whose face in contact with the first plate is etched so as to define microchannels between the two plates which are superimposed and glued to each other. The first plate can be made of a polymer material. The material constituting at least one of the two plates can be transparent. The dimensions of the microchannels are determined by adapting the width and the depth of the engravings in the engraved plate. It should be noted that microfluidic chips manufactured according to other methods known to those skilled in the art can obviously be used to implement the invention.

In addition to the elements mentioned above, the microfluidic circuit according to the invention can be connected in particular to at least one element chosen from an electronic device necessary for the operation of the system, a battery, a potentiostat for controlling the electrochemical measurements, a cooling system placed on the chips to cool the solutions coming from the tank and a touch screen in particular which makes it possible to launch the desired measurement, to know the progress of the measurement and to visualize the result obtained.

Microfluidic process

As indicated above, the invention relates to a microfluidic method for analyzing a fluid containing at least one ETM comprising the following steps:

c) measurement of the amount of ETM present in the sample obtained in b), using an electrochemical detection method.

b) mixing, within the microchannel of the microfluidic circuit, of the fluid sample introduced in step a), with nitric acid and L-cysteine, and c) measurement of the amount of arsenic present in the sample obtained in b), using an electrochemical detection method.

Step a) of introducing a fluid sample into at least one microchannel of a microfluidic circuit is preferably carried out according to the following substeps:

a1) injection of the sample into the microfluidic circuit; then

a2) pressurizing the sample in the circuit.

The injection of the sample into the microfluidic circuit (sub-step a1) is notably carried out by injecting said sample into the input of the first microchannel of the first chip of the circuit according to the invention. In particular, this step is carried out using a syringe fitted with a 0.45 μm filter. The filter removes all suspended solids with a diameter greater than 0.45 µm.

More specifically, with reference to the figure, initially the solenoid valves 9, 16 and 30 are open. The solenoid valve 9 allows the sample (E) to pass to the reservoir R.3. Part of this sample is used to rinse the tank and goes to the external PE bin through the solenoid valves 16 and 30, while the rest of the sample remains in the tank R.3 and is used for analysis.

Typically, this operation can last a few minutes or seconds, then the solenoid valves 9, 16 and 30 are closed.

Then, said sample is pressurized (sub-step a2). The sample can be pressurized by any means, for example by injection of a gas, in particular an inert gas, or by suction. For example, the pressurization can be carried out using a pump or a syringe. Thanks to its pressurization, the sample is set in motion.

For example, the sample is stored in a tank (R.3) connected to a microchannel of the microfluidic circuit. Preferably, it is stored in a reservoir (R.3) connected to the first microchannel of the first chip, and the pressurization of the reservoir R.3 is carried out in particular by the opening of a solenoid valve (the solenoid valve 3) , which is connected to the nitrogen gas bottle (G). Preferably, the other tanks, except tank R.3, are always under pressure during the whole process (ie before and after the measurement). The solenoid valve 3 remains open for the time of the analysis; it is closed at the end of the measurement.

Between steps a) and b) of the method according to the invention, step N) can be carried out: this is the cleaning step.

This step N preferably comprises at least one, preferably at least two, preferably at least three, preferably the following four substeps:

N1: a sub-step for cleaning the microfluidic circuit; and or

N2: a substep for cleaning the measurement electrodes, in particular the gold electrode; and or

N3: a sub-step of electrochemical gold deposition; and or

N4: a control sub-step, in particular by measuring a control solution.

Preferably, the sub-step N1 is carried out as follows:

The solenoid valves 1, 21 and 30 are open. The sample inlet channel in the pretreatment chip 50 (inlet 54 of the pretreatment chip) is cleaned with the sample, then the fraction of the sample used for cleaning is returned to the external bin, in particular to through the solenoid valves 21 and 30. This operation typically lasts about 30 seconds, then the solenoid valves are closed.

Thereafter, the solenoid valves 2, 5 and 29 are opened. The sample is pushed towards the tank, in particular through the solenoid valve 2, and conveyed to the internal bin, in particular thanks to the solenoid valves 5 and 29. This operation typically lasts 30 seconds, then the solenoid valves are closed.

Thereafter the tank is emptied, in particular using the solenoid valves 15, 13 and 30. The solenoid valve 15 sends the gas G (like the solenoid valve 3) into the tank; the gas then exerts a pressure on the liquid contained in the tank, and pushes it towards the external bin, in particular passing through the solenoid valves 13 and 30. This operation typically lasts 15 seconds, then the solenoid valves are closed.

Preferably, the sub-step N2 is carried out as follows:

Preferably, at least one electrode used in step c) of the method according to the invention, called the working electrode, is made of gold. In this case, it is preferable to clean it before any measurement, to eliminate potential oxides formed on its surface during time, or to eliminate the traces of ETM (arsenic in particular) remaining during the previous measurement on the electrode.

For this, sulfuric acid (contained in the tank R.12) is used to clean the gold electrode present in the main microchannel 69 of the analysis chip 60, in particular by cyclic voltammetry (voltammetry). The solenoid valves 18 and 29 are open to allow this acid to pass. Cycling is typically carried out between -0.4 and 1.5 V at 200 mV / s. This operation generally lasts 3 minutes, then the solenoid valves are closed.

Preferably, the sub-step N3 is carried out as follows:

The purpose of this sub-step N3 is to increase the electrochemically active surface of the gold, obtained by electrochemical deposition under vacuum.

The measurement surface will no longer be flat, but in relief (in 3D), because the electrochemical deposition leads to a non-planar surface. To do this, in general the solenoid valves 24 and 29 are open for approximately 3 minutes, the mixture of tetrachloroauric acid and sulfuric acid from the reservoir R.13 is then released in the inlet 74 then in the main microchannel 69, and the gold deposit is made by chronoamperometry for about 300 seconds at the Au (III) deposit peak potential on the working electrode. This potential is determined by cyclic voltammetry. The deposition of gold can also be carried out when after a certain number of measurements the active gold surface is reduced, which results in a reduction in the area of reduction peak of the gold oxides by measurement in voltammetry. cyclic. In this case, the system automatically launches a gold deposit to regenerate.

Preferably, the sub-step N4 is carried out as follows:

A measurement of the control solution (white) can be carried out to check the cleanliness of the previously cleaned circuit. For this, the solenoid valves 17 and 29 are generally open for about 5 minutes. The analysis of the blank (control solution) (contained in the tank R.5) is typically done by SW V (Square Wave Voltammetry, or square wave voltammetry in French).

The content of the reservoir R.5 is released in the inlet 62 and then in the main microchannel 69, to be measured, before being eliminated in the internal bin.

The analysis of the blank is done with a deposit potential (Edep) of -1.1 V, for 90 seconds (Tdep), with an amplitude of 0.02 V. The signal is recorded between -0.2 and 0, 7 V. If a peak appears, then the sub-steps N1 to N3 are restarted, preferably automatically, otherwise we go to step b) of the method according to the invention. Once the sample has been introduced, and the possible cleaning step (s) carried out, there follows the second step (step b): mixing, within the microchannel of the microfluidic circuit, of the sample with reagents, preferably nitric acid and L-cysteine.

Typically, during this step b), the two reagents in particular present in the two reservoirs fluidly connected to one end of the first chip are released in the first microchannel of said first chip, and mix with the sample injected into the inlet. .

Preferably, the mixture obtained is then conveyed in a tank, preferably in the tank connected to the second end of the first microchannel of the first chip.

Preferably, the first microchannel of the first chip is the main microchannel (microchannel 59 in FIG. 1), and has a width typically between 0.7 and 2 mm, preferably between 0.7 and 1.5 mm, preferably equal to 1 mm ; and a length typically between 30 and 60 μm, preferably between 40 and 55 μm.

Preferably, the flow rate of the microchannel of the first chip is 11 ml / h for a pressure of 500 mbar.

Typically, during this step b), the solenoid valves 4, 1, 5, 6, 12 and 29 are opened. The sample, the nitric acid and the L-cysteine are thus mixed in the desired proportions thanks to the chip. mixture 50 then routed into the tank through the solenoid valve 12. This operation generally lasts a few minutes, typically 2 to 5 minutes, preferably 2 to 3 minutes, more preferably 2 minutes and 15 seconds, then the solenoid valves are closed.

Then rinse the connection tubes and the tank with the pretreated sample.

As a result, the solenoid valves 1 1, 15 and 29 are opened, in particular for 15 seconds. The pretreated sample is thus sent to the internal bin.

The tank is filled again by opening the solenoid valves 4, 1, 5, 6, 12 and 29, generally for a few minutes, typically from 2 to 5 minutes, preferably from 2 to 3 minutes, more preferably 2 minutes and 15 seconds, then the pretreated sample is routed, this time to the analysis chip 60 by opening the solenoid valves 14, 15 and 29. Preferably, the dimensions of the different channels are chosen so that at the outlet of the main microchannel 59 of the chip 50, the mixtures are homogeneous and in specific proportions.

In particular, the mixture of sample, nitric acid (at 2.2 mM) and L-cysteine (at 50 mM) is produced in a respective volume ratio of 0.6-0.7: 0.03- 0.05: 0.25-0.40. Preferably, this respective volume ratio is equal to 0.63: 0.04: 032.

Finally, once the sample has been mixed with reagents, preferably nitric acid and L-cysteine, the method comprises a step c) of measuring the amount of ETM present in the sample obtained in b), using at least 3 electrodes, one of which is gold.

Step c) preferably includes the circulation of the sample obtained in b), from the tank through the second microchannel of the analysis chip, comprising at least three electrodes, one of which is made of gold.

Preferably, the second microchannel of the second chip (analysis chip) is the main microchannel (microchannel 69 in FIG. 1), and has a width typically between 0.1 and 2 mm, preferably between 0.12 and 1.5 mm; and a length typically between 5 and 80 mm, preferably between 9 and 60 mm.

Typically, during step c), the sample obtained in b) (also called pretreated sample), once in the analysis chip 60, is analyzed using at least 3 electrodes, one of which is made of gold.

Preferably, during step c), at least one standard solution is mixed with the sample obtained in b).

More specifically, in the case of arsenic, during step c), the pretreated sample is analyzed using at least 3 electrodes, one of which is made of gold, and according to the following substeps:

c1) measure the amount of arsenic (III) present in the sample, called As (III), then c2) convert the arsenic (V) remaining in the sample into arsenic (III), then measure the amount of arsenic (III) obtained, called As early, and finally

c3) determining the amount of arsenic actually present in the sample by the formula As (V) = As early - As (lll).

First, the amount of As (lll) is determined; this is step c1). Preferably, this step c1) involves at least one standard solution. Preferably, step c1) comprises mixing the sample obtained in b) with at least one standard solution, preferably two standard solutions of different concentrations, then determining the concentration of As (III) present in the sample. By "standard solution" is meant a solution comprising a known concentration of As (III). For example, it is possible to use a first standard solution comprising an As (III) concentration of between 5 and 15 ppb, and a second standard solution comprising an As (III) concentration of between 15 and 25 ppb; or it is possible to use a first standard solution comprising an As (lll) concentration of between 1 and 3 ppb, and a second standard solution comprising an As (lll) concentration of between 3 and 5 ppb

Preferably, the quantity of As (III) is measured by circulating the sample obtained in b) by SWV, in particular with the same electrochemical parameters as the control solution (white), with the exception of Tdep (deposition time ), i.e. a deposition potential (Edep) of -1.1 V, for 120 seconds (Tdep), with an amplitude of 0.02 V, and the signal is recorded between -0.2 and 0.7 V.

The mean of the area (Amoy) of the peak measured during the desorption of arsenic on the gold electrode is compared with threshold values.

In particular :

- if 3x10 ³ pAV <Amoy <3x10 ² pAV, then the system opens the solenoid valves 27 and 25, which provide concentrations of additions (standard solutions) in the mixture of 10 and 20 ppb respectively (ie tanks R.10 and R .1 1). With each addition, Amoy (addition) is measured. Thus, the values of Amoy, Amoy (addition 1) and Amoy (addition 2) are used to determine the concentration of As (III) in the sample;

- if Amoy <3x10 ³ pAV, then the Amoy measurement is repeated using a deposition time of 360 seconds. In this case, if the signal obtained gives an Amoy <3x10 ^-4 pAV, then the sample contains As (III) in concentration below the limit of quantification of 0.85 ppb. Conversely, if Amoy> 3x10 ^-4 pAV, then the system opens the solenoid valves 26 and 28, which provide concentrations of additions in the mixture of 2 and 4 ppb respectively (ie reservoirs R.8 and R.9) . With each addition, Amoy (addition) is measured. Thus, the values of Amoy, Amoy (addition 1) and Amoy (addition 2) are used to determine the concentration of As (III) in the sample; - if Amoy> 3x10 ² pAV, the sample is automatically diluted at the level of the pretreatment chip 50, and according to the measured Amoy. Then a Tdep of 360 seconds or 120 seconds is applied.

Then, the amount of As (V) is determined; this is step c2).

The measurement of As (V) is obtained indirectly by subtracting the actual As (lll) content from the total arsenic content (As early) (i.e. As (V) = As early - As (lll )). Total arsenic (early As) is obtained by converting all of the As (V) to As (lll).

To convert arsenic (V) to arsenic (III), preferably an incubation step of the sample obtained in b) takes place in the tank, preferably by heating. Typically, this incubation step is carried out for a few minutes.

For this, when the mixture is in the tank, the solenoid valves are closed, and the tank is heated using the heating resistor, in particular for 10 minutes. Thus all the As (V) is transformed into As (lll), which, combined with the As (lll) already present in the sample, gives the total amount of arsenic. This mixture is subsequently sent to the analysis chip 60 as previously described (i.e. by opening the solenoid valves 14, 15 and 29), to be dosed. The analysis chip 60, having a cooling system, makes it possible to cool the mixture around 22-23 ° C, then to carry out the measurement in As (III), in the same way as described above.

Finally, typically, after analysis, the solenoid valves 7, 12, 5 and 29 are open, in particular for 30 seconds, to clean the tank and the microchannels containing nitric acid. The nitric acid remaining in the tank is used to clean the analysis chip 60, typically by opening the solenoid valves 14, 15 and 29, for about 30 seconds. This chip is cleaned again, then filled with nitric acid for storage, generally for 30 minutes, by opening the solenoid valves 20 and 29.

The invention is now illustrated by the following example.

Example 1: implementation of the method according to the invention for measuring the arsenic level in a water sample

Analysis of arsenic-doped water samples was carried out. The choice fell on ulptrapure water and two mineral waters (Volvic and Evian). Before analysis, these waters are analyzed beforehand by ICP-MS to ensure that they do not contain arsenic, or that the amount of arsenic contained is less than the limit of quantification of the instrument used (0 , 1 ppb). Subsequently, these waters to be analyzed are doped with 10 ppb of As (III) to obtain an ultrapure water containing 10 ppb of As (III) and two samples of mineral water each containing 10 ppb. The choice of 10 ppb of As (III) is relative to the WHO standard which fixes the arsenic thresholds in drinking water at 10 ppb. As (lll) is chosen in this test because it is the most toxic form of arsenic. For the analysis, 40 mL of As (III) water sample is injected using a syringe into the microfluidic analysis system according to the invention. The method according to the invention is implemented for the analysis of these samples in turn on the glass chip. The response of the sensors is recorded in triplicate for each sample and the results below are obtained:

The arsenic introduced is almost completely detected by the method described here.

In conclusion, the method according to the invention can be used effectively for the automatic detection of ETM in water.

Claims

1. Microfluidic method for analyzing a fluid containing at least one metallic trace element comprising the following steps:

c) measurement of the quantity of metallic trace element present in the sample obtained in b), using an electrochemical detection method,

said step c) comprising the mixing of the sample obtained in b) with at least one solution comprising a known concentration of metallic trace element, then the assay of the metallic trace element by electrochemical detection method.

2. Microfluidic method according to claim 1, characterized in that the metallic trace element is arsenic, and in that the method comprises the following steps:

c) measuring the amount of arsenic present in the sample obtained in b), using an electrochemical detection method, preferably at least 2 electrodes, preferably at least 3 electrodes, one of which is made of gold.

3. Microfluidic method according to claim 1 or 2, characterized in that the measurement of step c) is carried out using the following three electrodes:

- a gold electrode, as a working electrode,

- a platinum electrode, as a reference electrode, and

- a platinum counter electrode.

4. Microfluidic method according to any one of the preceding claims, characterized in that step a) is carried out according to the following substeps:

a1) injection of the sample into the microfluidic circuit; then

a2) pressurization of the sample in the circuit.

5. Microfluidic method according to one of claims 2 to 4, characterized in that, between steps a) and b), a cleaning step N) is carried out and preferably comprises at least one, preferably at least two, preferably at least three, preferably the following four substeps:

N1: a sub-step for cleaning the microfluidic circuit; and or

N2: a substep for cleaning the electrodes, in particular the gold electrode; and or

N3: a sub-step of electrochemical gold deposition; and or

N4: a control sub-step, in particular by measuring a control solution.

6. Microfluidic method according to any one of the preceding claims, characterized in that the microfluidic circuit comprises:

- at least one reagent storage tank (s), preferably nitric acid and L-cysteine and optionally at least one second storage tank comprising at least one standard solution;

o at a first end, two tanks and an inlet, and o at the second end, a tank,

said input suitable for injecting the sample; and

7. Microfluidic method according to claim 6, characterized in that it comprises two storage tanks for two separate reagents and in that, during step b), the two reagents present in the two tanks connected to one end of the first chip are released in the first microchannel of said first chip, and mix with the sample injected into the inlet, and preferably the mixture obtained is then conveyed in a tank, preferably in the tank connected to the second end of the first microchannel of the first chip.

8. Microfluidic method according to any one of claims 2 to 7, characterized in that the mixture of sample, nitric acid used at 2.2 mM and L-cysteine used at 50 mM from step b) is carried out in a respective volume ratio of 0.6-0.7: 0.03-0.05: 0.25-0.40, preferably this respective volume ratio is equal to 0.63: 0.04: 032 .

9. Microfluidic method according to any one of claims 2 to 8, characterized in that step c) comprises:

10. Microfluidic method according to any one of claims 1 to 9, characterized in that step c) comprises mixing the sample obtained in b) with at least two solutions each comprising a known concentration of metallic trace element , then the metering of the metallic trace element by electrochemical detection method.

1 1. Microfluidic method according to claim 10, characterized in that the two solutions each comprising a known concentration of metallic trace element have a concentration of less than 10 ppb, for example 2 and 4 ppb; or a concentration greater than or equal to 10 ppb, for example between 10 and 20 ppb, for example at 10 and 20 ppb.

12. Microfluidic method according to claim 1 1, characterized in that the determination of the metallic trace element only comprises its detection in the range of concentrations less than 10 ppb or in the range of concentrations greater than or equal to 10 ppb.

13. Microfluidic circuit for analyzing a fluid, in particular capable of implementing the method according to one of claims 2 to 12, comprising:

- at least two storage tanks for nitric acid and L-cysteine;

- at least a first chip, called a premix chip, comprising at least a first microchannel fluidly connected:

o at a first end, two tanks and an inlet, and o at the second end, a tank,

said inlet suitable for injecting a sample of fluid to be analyzed; and

- at least a second chip, called an analysis chip, comprising at least a second microchannel connected to the tank and comprising at least three electrodes, one of which is made of gold.